Cell Cycle Analysis in Fixed Cells

Multicolor cell cycle studies are possible using flow cytometry and it is advantageous to analyze DNA content on alternative lasers to preserve the common 488 nm laser for other markers.  This flexibility can be applied to cell cycle analysis using FxCycle Violet, SYTOX AADvanced, FxCycle PI/RNAse Solution, or FxCycle Far Red stains on the violet, blue, or red lasers, respectively. These products:

  • Allow increased flexibility for multicolor cell cycle studies
  • Require little or no compensation with 488 nm excitable dyes 
  • Result in tight CVs for more accurate analysis

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Violet laser—fixed cell cycle analysis

FxCycle Violet

FxCycle Violet Stain is a violet laser–excited dye used for cell cycle analysis in fixed cells. Using FxCycle Violet for cell cycle analysis increases the ability to multiplex by freeing up the 488 nm and 633 nm lasers for other cellular analyses such as immunophenotyping, apoptosis analysis, and dead cell discrimination.

Because of its narrow emission spectra, FxCycle Violet Stain overlaps less with other fluorescent channels than other commonly used dyes (propidium iodide and 7-AAD), resulting in minimal compensation requirements and more accurate data.

FxCycle Violet Stain
Figure 1. Multiparametric cell cycle and immunophenotypic analysis. TF-1 erythroblast cells were alcohol-fixed overnight, washed, and then suspended in 0.1% Triton™ X-100/PBS/1% BSA before staining with anti–histone H3[pS10] purified antibody complexed with Zenon Alexa Fluor 488 Rabbit IgG labeling reagent and FxCycle Violet stain. The pH3 signal (red) identifies cells that are in mitosis.

Blue laser—fixed cell cycle analysis

SYTOX AADvanced Dead Cell Stain

SYTOX AADvanced Dead Cell Stain is a new high-affinity nucleic acid stain for the detection of dead cells and analysis of cell cycle using the common 488 nm blue laser in flow cytometry. The dye is spectrally similar to 7-AAD but with rapid kinetics of uptake and relatively low CVs.

SYTOX AADvanced Dead Cell Stain penetrates cells more efficiently than 7-AAD providing better separation of live and dead cells. SYTOX AADvanced Dead Cell Stain can also be used with fixed cells for DNA content analysis when paired with RNAse treatment. SYTOX AADvanced Dead Cell Stain Kits contain a vial(s) of dried dye and anhydrous DMSO providing stable shelf life.

SYTOX AADvanced Dead Cell Stain
Figure 2. A mixed population of Jurkat cells were stained with SYTOX AADvanced Dead Cell Stain Kit and analyzed by flow cytometry. A mixture of heat-killed and untreated Jurkat cells were stained with 1uM SYTOX AADvanced Dead Cell Stain Kit for 5 minutes. Cells were analyzed on a flow cytometer equipped with a 488 nm laser and a 695/40 nm bandpass filter. Live cells are easily distinguished from the dead cell population.

FxCycle PI/RNAse Solution

FxCycle PI/RNase Staining Solution is used for flow cyotmetric analysis of DNA content in fixed cells. Propidium iodide (PI), a popular red-fluorescent stain, binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. PI also binds to RNA, necessitating treatment with RNase to distinguish between RNA and DNA staining. This propidium iodide solution includes DNase-free RNase A and a permeablization reagent. It comes ready to use: simply add the staining solution to fixed cells, incubate, and acquire on a flow cytometer without washing.

FxCycle PI/RNase Staining Solution
Figure 3. Histogram of Jurkat cells stained with FxCycle PI/RNase stain showing DNA content distribution. Jurkat cells were fixed in 70% ethanol, washed, and then resuspended in FxCycle PI/RNase stain for 30 minutes at room temperature. G0/G1 and G2/M phase histogram peaks are separated by the S phase distribution. Analysis was performed using 532-nm excitation with a 585/42-nm bandpass filter.

Red laser—fixed cell cycle analysis

FxCycle Far Red

FxCycle Far Red stain is used for flow cytometric analysis of DNA content in fixed cells when combined with RNAse. This dye takes advantage of the commonly available 633⁄5 nm excitation sources with emission around 660 nm.

With multicolor cell cycle studies possible using flow cytometry, it is often necessary to analyze DNA content on alternative lasers to preserve the common 488 nm laser and detection channels for other markers. Well suited for the popular red laser, FxCycle Far Red stain is a good choice for DNA content analysis in multicolor cell cycle studies.

FxCycle Far Red stain
Figure 4. Histogram of TF-1 erythroblast cells stained with FxCycle Far Red stain showing DNA content distribution. TF-1 cells were fixed overnight with alcohol, washed, and then resuspended in 0.1% Triton™ X-100/PBS/1% BSA before staining with FxCycle Far Red stain plus RNase A for 30 minutes at room temperature. G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution. Analysis was performed using 633 nm excitation with a 660/20 bandpass filter.

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